Cell suspension preparation technique and device

ABSTRACT

The present invention provides for methods and devices suitable for producing a transplantable cellular suspension of living tissue suitable for grafting to a patient. In applying the method and/or in using the device, donor tissue is harvested, subjected to a cell dissociation treatment, cells suitable for grafting back to a patient are collected and dispersed in a solution that is suitable for immediate dispersion over the recipient graft site.

RELATED APPLICATIONS

The present invention is a divisional application of U.S. patentapplication Ser. No. 13/036,569, filed Feb. 28, 2011, which is acontinuation of U.S. patent application Ser. No. 10/068,299, filed Feb.6, 2002, which claims priority to and the benefit of AustralianProvisional Patent Application PR2989, filed Feb. 7, 2001 and U.S.Provisional Patent Application No. 60/281,527, filed Apr. 4, 2001, thedisclosures of all of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

This invention relates to a simple, rapid and cost effective techniquefor grafting of cells, and in particular to a device for preparing asuspension of cells from a tissue sample obtained from a donor site andapplying that suspension of cells to a recipient site.

BACKGROUND ART

There are many methods of treating wounds known to those skilled in theart. For example, skin grafting techniques exist which aim toreconstruct the skin covering areas of the body where there is eitherdamage or defects to the skin. In general, these types of grafts areclassified according to their host-donor relationship and by theirthickness. The most clinically applied graft is the autologous graft,whereby tissue is taken from one area of the body and applied to anotherarea. The grafted tissue then develops a new blood supply and attachesto the underlying tissues.

There are several types of skin grafts presently used, includingsplit-thickness, full-thickness grafts, and micro-grafting. Each ofthese graft types must be prepared using certain techniques, and eachone has its inherent advantages and disadvantages. Split-thicknessgrafts often require considerable skill, time and expensive equipment toperform. Further, donor sites are painful, result in scarring and limitthe area able to be covered. Although they may be more successful thanfull-thickness grafts, they are usually less cosmetically attractive.Full-thickness grafts require less skill or expensive equipment, andtheir cosmetic appearance is better than that of split-thickness grafts.However, full-thickness grafts do not “take” as well as split-thicknessgrafts. Micro-grafts are more easily accomplished and require no specialinstruments. However, their cosmetic appearance is not as good as othertechniques, as the resulting scarring is unacceptable.

A variation to the above grafting techniques is the mesh graft, which isa type of split-thickness or full-thickness skin graft in which parallelrows of slits are cut in the tissue being treated. Some of theadvantages of mesh grafts include: a greater coverage of the effectedarea, drainage of blood or serum from under the graft, and increasedconformity of the graft to uneven recipient areas. This technique hasbeen very successful, with 90 to 100 percent “take” when the grafts havebeen applied on healthy granulation beds.

An alternative to split-skin grafting is to form a blister under suctionat a donor site and transplant to the recipient site. The production ofblisters to treat wounds has been used since the 1960. The blisters areproduced by a suction device, such as Dermavac™, at a suction pressureof approximately 250-300 mmHg for 1-2 hours. The blisters are then cutoff and placed on the wound. The healing time is around 10-14 days.There are several disadvantages of this method such as the amount oftime required to prepare the graft is too long and the graft may notresult in re-pigmentation of the area; or uneven pigmentation is commonaround the edges of the area of treatment.

Micro-grafting has become a more common approach for large area coverand involves the “snipping off” of a number of very small sections oftissue from a donor site and applying then to a dressing, which is inturn applied to the wound area.

The most advanced technology for the generation of a tissue in vitro isto culture epidermis. Cultured epithelial autografts (CEA) are animportant adjunct in the coverage of burns and other situations in whichlarge areas of the body's surface experience skin loss. There are manycenters throughout the world with tissue culture facilities whose aim isto produce autologous epithelial grafts for use in a wide variety ofapplications. The usefulness and application of CEA is related to itsability to achieve confluent cells sheets suitable for grafting. Thistechnique overcomes many of the disadvantages of the previous treatmentsdescribed above. For example, cultured epithelial autografts reduce thedemand for donor sites. However, these autografts are slow growing andrequire time for culturing of the grafts, which often exceeds the timeof preparation of the recipient's sites.

The present invention provides a cellular suspension together with amethod for preparing that suspension and a device for its preparationeach of which seek to ameliorate one or more of the disadvantagesassociated with prior art grafting technology.

SUMMARY OF THE INVENTION

The subject invention relates to a unique cell suspension and method forits preparation that is rapid, efficient and simple to prepare andapply. It also relates to a method for treating a patient using theunique cell suspension and to an apparatus suitable for use in themethod preparation. Use of the described device while not essential forpracticing the method of the invention has been found to significantlyreduce the complexity associated with the use of conventional graftingtechnology such as CEA.

According to a first aspect of the invention there is provided a methodfor preparing a cell suspension suitable for application to a patient,which method comprises the steps of:

(a) subjecting a tissue sample including cells suitable for grafting toa patient, to at least a physical and or chemical dissociating meanscapable of dissociating cellular stratum in the tissue sample;

(b) removing the tissue sample from the presence of the tissuedissociating means used in step (a) and harvesting in the presence of anutrient solution cells from the tissue sample, cells suitable forgrafting on to a patient wherein the nutrient solution is (i) free ofxenogenic serum, (ii) capable of maintaining the viability of the cellsuntil applied to a patient and (iii) is suitable for direct applicationto a region on a patient undergoing tissue grafting; and

(c) filtering the cellular suspension produced according to step (b) toremove large cellular conglomerates.

In a preferred form of the invention the dissociating means is of achemical nature such as an enzyme which is capable of disruptingcellular bonding like for example trypsin. Further, preferably thefiltered cellular suspension is diluted to an appropriate cell densityusing a nutrient solution, which may be anything from a basic saltsolution to a more complex nutrient solution.

According to a second aspect of the invention there is provided a cellsuspension produced according to the above method. Preferably the cellsin the suspension are autologous cells (i.e. they are isolated from thepatient requiring an autograft). The inventors have observed that byremoving xenogenic serum from the cell suspension there is lesslikelihood of transmission of infection and xenogenic reactions betweena patient and the serum are eliminated. Another feature of the cellsuspension produced according to the above method is that the tissuesample used to isolate the cells in the suspension is removed from theenzyme solution before the cells are harvested. When cells are exposedto enzymes capable of damaging inter-cellular adhesion the viability ofthe cellular suspension decreases over time thereby reducing theefficiency of the grafting when applied to a patient. The cellsuspension produced according to the above method has been observed topossess greater cellular viability compared to comparative methods thatharvest the cells at regular intervals whilst the tissue is immersed inthe presence of enzymes like trypsin.

According to a third aspect of the invention there is provided a methodof treating a patient in need of graft surgery, said method comprisingthe steps of:

(a) preparing an cell suspension according to the above method;

(b) administering the suspension directly to a region on the patientthat requires a cell graft in a manner that facilitates spreading of thecell suspension in a relatively even distribution over the graft region.

In an alternate form, the invention consists in the use of a cellularsuspension suitable for grafts, which suspension is prepared accordingto the following steps:

(a) subjecting a tissue sample including cells suitable for grafting toa patient, to an enzyme suitable for dissociating cohesive pieces of thetissue stratum in the sample;

(b) removing the sample from the enzyme solution used in step (a) andharvesting in the presence of a nutrient solution cells from the tissuesample, which cells are suitable for grafting on to a patient whereinthe nutrient solution is (i) free of xenogenic serum, (ii) capable ofmaintaining the viability of the cells until applied to a patient and(iii) is suitable for direct application to a region on a patientundergoing tissue grafting;

(c) filtering the cellular suspension produced according to step (b) toremove large cellular conglomerates;

for the preparation of medicament suitable for the treatment of tissuedisorders requiring grafting.

According to a fourth aspect of the invention there is provided anapparatus for developing a tissue regeneration solution, said apparatuscomprising:

(a) a heating means suitable for heating an enzyme solution to arequired temperature and which is capable of maintaining that solutionat the desired temperature for a suitable amount of time; and

(b) a filter recess comprising a filter means capable of separatinglarge cellular congregates from a cellular suspension.

When the enzyme solution is placed in contact with the heating means itis preferably heated in a manner that avoids localized heating withinthe solution.

Other aspects and advantages of the invention will become apparent tothose skilled in the art from a review of the ensuing description, whichproceeds with reference to the following illustrative drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a perspective view of the apparatus with the lid openand the second member in place.

FIG. 2 illustrates a perspective view of the apparatus with the lid openand the second member removed and inverted.

FIG. 3A illustrates a perspective view of the first member of theapparatus.

FIG. 3B illustrates a perspective rear view of the first member of theapparatus.

FIG. 4 illustrates a perspective view of the base of the apparatus.

DETAILED DESCRIPTION OF THE INVENTION

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variation and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in the specification, individually or collectively andany and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally equivalent products, compositions andwhere appropriate methods are clearly within the scope of the inventionas described herein.

Throughout this specification and the claims that follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

Having regard to the above, this invention provides a unique method andor device suitable for producing a transplantable cellular suspension ofliving tissue suitable for grafting to a patient. In applying the methodand/or in using the device donor tissue is harvested, subjected to atissue dissociating means, cells suitable for grafting back to a patientare collected and dispersed in a solution that is suitable for immediatedispersion over the recipient graft site.

The subject invention has many advantages over the prior art some ofwhich are illustrated in the following paragraphs.

1. It provides a time efficient method for supplying a cellular cover toa tissue in a clinical setting. That is, cells are available when neededat the time of surgery. This is achievable because there is a very shortpreparation period of the cells, thus allowing grafting to be performedperi-operatively or in the rooms of a specialist physician or GeneralPractitioner.

2. It provides a method and an apparatus which significantly reduces thecomplexity associated with the use of conventional CEA's and isparticularly useful in cases of burn injury that have presented late. Insome instances, cells are unavailable at the time of surgery, either dueto delayed referral of a patient with an unhealed burn or simply becausethe time needed for culturing of the grafts had exceeded that forpreparation of the recipient would bed. The present inventionameliorates the issue of graft preparation time.

3. It aids in the achievement of rapid cell coverage in areas of injuryand donor sites. It provides a means for reducing the size of donorsites—the biopsy donor site is markedly smaller than a split skin graftdonor site and reduces or eliminates the use of split skin graft donorsites; improves expansion rate of cell coverage; improves the rate ofhealing of small burns; is useful for small areas of skinreconstructions, such as scars; and improves scar quality.

4. It ameliorates problems associated with the use of solutions usedduring conventional tissue culture process. According to the method ofpreparation and treatment the cells used in a graft are suspended in anutrient solution free of xenogenic serum. That suspension is thenplaced directly onto the recipient site.

5. It provides a means for the treatment of various skin disorders ordiseases. For example, it may be used for the following: epidermalresurfacing, replacement after skin loss, site match-up duringre-pigmentation of an area of skin, treatment of burn wounds,leukoderma, vitiligo, piebaldism, in the treatment of scars—for example,caused through incorrect wound healing, improper scar direction or scardistortion from wound contraction, acne scars; resurfacing cosmeticdermabrasion, resurfacing after laser treatment and in association withdermal reconstruction. Additionally the method may be used for cellreplacement therapy, including, for example, nerve cell replacementtreatment, epithelial cell (such as urothelial cell, buccal mucosal celland respiratory epithelial cell) replacement treatment, endothelial cellreplacement treatment and osteogenic precursor cell replacementtreatment.

6. It provides a means to produce a suspension of cells in a ratio toeach other comparable with those seen in situ. That is, due to themanner of preparation of the cellular suspension, cells such askeratinocyte basal cells, Langerhans cells, fibroblasts and melanocytestypically have enhanced survival rates in comparison to standard tissueculture techniques, whereby selective cell culture can result in theloss of certain cell types. This has the advantage of allowing for thecorrect re-pigmentation of skin after a skin graft.

7. It allows faster surgery and healing—thereby reducing trauma forpatients during the phase of their medical care.

According to the first aspect of the invention there is provided amethod for preparing a cell suspension suitable for use in resurfacingand regeneration of damaged tissue.

According to this method, tissue (preferably of an autologous nature) isharvested from a patient by means known in the art of tissue grafting.Preferably this is achieved by taking a tissue biopsy. With theharvesting of the biopsy consideration must be given to the depth of thebiopsy and the surface area size. The depth and size of the biopsyinfluence the ease at which the procedure can be undertaken and speedwith which a patient recovers from the procedure. In a highly preferredform of the invention the donor site should be chosen to appropriatelymatch the recipient site, for example post-auricular for head and neck,thigh for lower limbs, inner-upper-arm for upper limbs, or palm for soleor vice-versa.

Once a biopsy has been harvested from a patient the tissue sample issubjected to physical and/or chemical dissociating means capable ofdissociating cellular stratum in the tissue sample.

Methods for dissociating cellular layers within the tissues are wellknown in the field. For example, the dissociating means may be either aphysical or a chemical disruption means. Physical dissociation meansmight include, for example, scraping the tissue sample with a scalpel,mincing the tissue, physically cutting the layers apart, or perfusingthe tissue. Chemical dissociation means might include, for example,digestion with enzymes such as trypsin, dispase, collagenase,trypsin-EDTA, thermolysin, pronase, hyaluronidase, elastase, papain andpancreatin. Non-enzymatic solutions for the dissociation of tissue canalso be used.

Preferably, dissociation of the tissue sample is achieved by placing thesample in a pre-warmed enzyme solution containing an amount of enzymesufficient to dissociate cellular stratum in the tissue sample. This maybe achieved, for example using a trypsin solution, however, any otherenzyme such as dispase, collagenase, trypsin-EDTA, thermolysin, pronase,hyaluronidase, pancreatin, elastase and papain that cause cells tobecome detached from other cells or from solid surfaces may be used forthis purpose. When the enzyme used is trypsin the enzyme solution usedin the method is preferably calcium and magnesium free. One suchsolution is preferably calcium and magnesium ion free phosphate bufferedsaline.

Where the tissue biopsy is derived from a patient's skin (comprisingepithelial-dermal cells) the amount of trypsin that might be used in themethod is preferably between about 5 and 0.1% trypsin per volume ofsolution. Desirable the trypsin concentration of the solution is about2.5 to 0.25%, with about 0.5% trypsin being most preferred.

The time period over which the tissue sample is subjected to the trypsinsolution may vary depending on the size of the biopsy sample taken.Preferably the tissue sample is placed in the presence of the trypsinsolution for sufficient time to weaken the cohesive bonding between thetissue stratum. For example, where the tissue sample is taken from apatient's skin the sample might be placed in trypsin for a 5 to 60minute period. Preferably, the tissue sample is immersed in the trypsinsolution for between 10 and 30 minutes with 15 to 20 minutes beingoptimal for most tissue samples.

After the tissue sample has been immersed in the trypsin solution for anappropriate amount of time, the sample is removed from the trypsin andwashed with nutrient solution. Washing the tissue sample may involveeither partial or complete immersion of the treated sample in thenutrient solution. Alternatively, and more preferably, the wash solutionis dripped on the tissue sample in sufficient volume to remove and orsignificantly dilute any excess trypsin solution from the surface of thesample. Preferably any dilution that might occur would lead to less than0.05% trypsin in the nutrient solution.

The nutrient solution used in the method should be capable ofsignificantly reducing and more preferably removing the effect of thetrypsin either by dilution or neutralization. The nutrient solution usedin the method will also preferably have the characteristics of being (i)free of at least xenogenic serum, (ii) capable of maintaining theviability of the cells until applied to a patient, and (iii) suitablefor direct application to a region on a patient undergoing tissuegrafting. The solution may be anything from a basic salt solution to amore complex nutrient solution. Preferably, the nutrient solution isfree of all serum but contains various salts that resemble thesubstances found in body fluids; this type of solution is often calledphysiological saline. Phosphate or other non-toxic substances may alsobuffer the solution in order to maintain the pH at approximatelyphysiological levels. A suitable nutrient solution that is particularlypreferred is Hartmann's solution.

After application of the nutrient solution to the tissue sample, thecellular stratum of the sample are separated permitting cells capable ofreproduction to be removed from the cellular material and suspended inthe nutrient solution. Where the tissue sample is skin the dermis andepidermis are preferably separated to allow access to thedermal-epithelial junction of both surfaces.

Cells capable of reproduction are then removed from the separatedstratum by any means known in the art. Preferably, the reproductivecells are scraped off the surface of the stratum using an instrumentsuch as a scalpel. Cells capable of reproduction within thedermal-epithelial junction include but are not limited to keratinocytebasal cells, Langerhans cells, fibroblasts and melanocytes. Followingrelease of the cells from the tissue sample they are suspended in thenutrient solution. Preferably only a small volume of nutrient solutionis applied to the tissue sample during this harvesting step otherwisethe suspension may become too fluid therein providing difficulties inapplying the suspension to the graft.

To avoid excessively large cellular congregates in the cellularsuspension the suspension is preferably filtered. Any filter capable ofseparating excessively large cellular congregates from the suspensionmay be used in this preferred step of the invention. In a highlypreferred form of the invention the filter size is between 50 μm and 200μm. More preferably it is between 75 μm and 150 μm, with 100 μm beingone specific example.

Prior to application to the graft site or immediately after filtering,the cellular suspension may be diluted to produce an appropriate celldensity suitable for the purpose to which the suspension is to be used.

According to the second aspect of the invention there is provided anaqueous cell suspension produced by the method described in the firstaspect of the invention. The cell suspension provided by this method ishighly suitable for tissue regeneration and grafting techniques. Animportant advantage of utilizing such a suspension in graftingtechnology is that it can be used to greatly expand the area or volumeof a wound that can be treated quickly by in situ multiplication of alimited number of cells. The number and concentration of cells seededonto graft site may be varied by modifying the concentration of cells insuspension, or by modifying the quantity of suspension that isdistributed onto a given area or volume of the graft site.

By suspending cells in a nutrient solution which is at least (i) free ofxenogenic serum, (ii) capable of maintaining the viability of the cellsuntil applied to a patient and (iii) is suitable for direct applicationto a region on a patient undergoing tissue grafting, the inventors havefound that the outcome of patient grafts is improved. A partialexplanation for this appears to be attributable to the removal ofxenogenic serum and more preferably all serum from the cell suspension.Xenogenic serum is a common additive in grafting culture medium and iswell known to cause potential infective and hypersensitivity problems.Such serum is however generally required for the in vitro expansion ofthe cells and to neutralize the action of the enzyme if the enzyme usedis trypsin. The nutrient solution used in the present invention does notrequire such serum because the cell population within the suspension isnot expanded prior to application to the graft site. Rather cellularmultiplication is encouraged on the patient rather than in an in vitrosystem. When trypsin is used neutralization is achieved by other means.

Another unique feature of the cell suspension produced according to themethod of the first aspect of the invention is that the composition ofcells in the cellular preparation is comparable to that seen in situcompared to prior art cellular preparation. One possible explanation forthis is that in the prior art, culture of the cellular preparationutilizes selective culture for keratinocytes, therefore loss of cellularconstituents such as fibroblasts and melanocytes occurs whereas thecellular suspension produced from the first aspect of the invention hasa cell composition comparable to the in situ cell population. Anotherfeature of the cellular suspension produced from the first aspect of theinvention is that the graft cells are more viable as they are harvestedin a nutrient solution as distinct from prior art cell harvestingprocedures which utilize techniques where the cells are harvested whilstexposed to powerful digestive enzymes for excessive periods of time.When the cells are exposed to such enzymes for excessive periods of timethe viability of the cellular suspension decreases.

According to the third aspect of the invention there is provided amethod of treatment of the patient requiring a tissue graft. By thismethod the cellular suspension produced according to the first aspect ofthe invention is applied to a graft site. A liquid suspension containingcells may be manually distributed onto the graft site by any of severaltechniques, which include spraying, spreading, pipetting and painting.

In a highly preferred form of the invention the suspension is sprayed onto a graft site. The suspension may be sprayed through any type ofnozzle that transforms liquid into small airborne droplets. Thisembodiment is subject to two constraints. First, it must not subject thecells in solution to shearing forces or pressures that would damage orkill substantial numbers of cells. Second, it should not require thatthe cellular suspension be mixed with a propellant fluid that is toxicor detrimental to cells or wound beds. A variety of nozzles that arecommonly available satisfy both constraints. Such nozzles may beconnected in any conventional way to a reservoir that contains thecellular suspension.

Alternatively the suspension may be delivered via a pipette, common“eye-droppers,” syringe and needle and or other similar devices to placesmall quantities of cellular suspension on a graft site.

After the cell suspension has been applied to the recipient graft site,the wound may be dressed with a wound dressing. In a preferredembodiment the dressing is Surfasoft™ a woven nylon dressing.Preferably, the healing of the wound is followed up by standardprotocols for skin graft treatment known to those skilled in the art.

According to the fourth aspect of the invention there is provided anapparatus for developing a tissue regeneration solution, which has aheating means suitable for heating an enzyme solution to a requiredtemperature and which is capable of maintaining that solution at thedesired temperature for a suitable amount of time; and a filter recesscomprising a filter means capable of separating large cellularcongregates from a cellular suspension.

In a preferred form of the fourth aspect of the invention the apparatusalso includes a reservoir capable of holding a tissue sample and anutrient solution which solution is also capable of maintaining theviability of the cells in the tissue sample. More preferably thereservoir is of sufficient size to permit manipulation of the tissuesample permitting separation of the tissue cellular stratum andharvesting of those cells from the stratum suitable for grafting.

The apparatus may also include one or more fluid containment wells forstorage of fluids such as the nutrient solution. The wells mayalternatively serve as a receptacle for a container such as a plastic orglass vial that holds the nutrient solution. Preferably, the well iscapable of holding at least a 10 ml volume. Such wells permit ease offluid application to the tissue sample. Storage of such fluids in closeproximity to its site of application also provides the advantage ofreducing the risk of accidental leakage of the fluid and provides aneasy means for accessing the fluid for accurately delivering it toeither the tissue sample or the cell suspension.

In a highly preferred form of the invention the apparatus comprises afirst and second member wherein:

(1) the first member includes:

(a) at least a heating means suitable for heating an enzyme solution toa required temperature and which is capable of maintaining that solutionat the desired temperature for a suitable amount of time;

(b) at least a filter recess comprising a filter means capable ofseparating large cellular congregates from a cellular suspension;

(c) at least a fluid containment well for storage of nutrient solution;

(2) the second member forms a reservoir capable of withholding a tissuesample and nutrient solution in fluid containment; and

wherein the first member provides a seat upon which the second membermay be placed during manipulation of the tissue.

In a further preferred form of the invention the first member providesan storage compartment into which tools and solutions used in the abovedescribed method may be stored. Where such a compartment is provided inthe apparatus the second member may provide the lid or closure to thatcompartment. In use the lid is preferably removed from the top of thecompartment and inverted. The underside of the lid preferably forms thereservoir therein enabling the second member to serve a dual purpose.Tools and solutions used in the method can be accessed from thecompartment. The inverted lid may then be seated back over thecompartment therein providing the reservoir for the apparatus.

The apparatus may be made from metal, plastic or any other material.Further, the container may be any size. Preferably the size of thecontainer is only limited by its intended use and the need forsterilization such as by the use of gamma irradiation and or ethyleneoxide.

It should be appreciated that the heating means employed in theapparatus may simply constitute a heating pad or pads on the top of thefirst member. There are however, attendant problems with sucharrangements, not least of which is the possibility of accidentalspillage of the container undergoing heating. Therefore, in anembodiment, one or more heating means may be housed within a recess inthe first member. Also located within that recess is at least acontainer into which tissue may be placed for exposure to the enzymesolution. In an alternate embodiment one or more heating means may behoused in the base of the apparatus. In such a configuration the firstmember contains at least an opening suitable for receiving a containercapable of holding fluid, which opening provides access for thecontainer to the heating means.

It will be appreciated that if the apparatus is designed for more thanone use the heating means may be capable of being repeatedly heated andcooled. Alternatively each heating unit may be capable of a single use,but multiple heating units may be provided with the apparatus tofacilitate multiple heating events.

In a highly preferred configuration of the apparatus a heating collar islocated within a recess therein forming a heating recess in the firstmember within which there is located a container (e.g., a vial) for theenzyme. The container is preferably held in place by at least arestraining means, which desirably surrounds part of the upper portionof the container preventing accidental release of the container from theapparatus. In circumstances, where the apparatus is intended for singleuse the restraining means may be formed as an integral part of the firstmember, thus meaning that removal of the container may only be achievedby physically breaking the first member.

The heating means used in the apparatus is preferably controlled bycircuitry permitting activation of the heating element when required.For example the heating means may be switched on by depressing the startbutton located, for example, on the surface of the first member.Alternately the heating means may be activated by pushing the containerdown with sufficient force to activate a switch located in the base ofthe apparatus. A person of ordinary skill in the field will appreciatethat a wide range of electronic means may be used to activate theheating unit provided in the apparatus.

Desirably the heating unit is also operably linked to a timer mechanism,which is adapted to heat the enzyme solution for a pre-defined period oftime. In circumstances where the apparatus is intended for multipleuses, preferably the timer can be set to deactivate the heating elementwhen a particular amount of time is reached. At which point an alarm mayactivate to inform the user that the time is up. The alarm may beaudible or in the form of a light display.

In a further preferred embodiment of the present invention the heatingmeans may be provided with an adjustable temperature control. Wheretemperature adjustment is required such variation may be achieved byadapting the heating control circuitry to include or communicate with atemperature control mechanism permitting the temperature of the heatingunit to be constantly varied within a constant range, or it may presenta range of select temperatures that the heating control means can be setat. A temperature control means will beneficially be included in theapparatus where the apparatus is to be used in the harvesting andpreparation of different cell types and or where different enzymes areused in the harvesting method for which the apparatus has uniqueapplication.

In an alternate more preferred form, the apparatus is designed forsingle use. In such instances the timer mechanism is part of thecircuitry that controls the heating means. Once the heating means hasbeen activated it heats the solution for a predefined period of time andthen self-destructs. It should be appreciated by those skilled in theart that such an the apparatus may be fitted with various monitoringmeans that are capable of indicating such things as: the enzyme hasreached the required temperature; the amount of time that the enzyme hasbeen in the solution for; and or the amount of time left before thecircuitry self-destructs etc. By way of example only, the monitoringmeans might consist of a series of LED's that activate when certainevents occur. In a highly preferred embodiment the heating elementpreferably remains in the heating mode for a maximum of 45 minutes to 1hour.

The heating means may be powered by any means known in the art.Preferably, the power supply is provided by battery/batteries. In oneform of the invention, the power supply is a battery or a plurality ofbatteries located in the base of the apparatus.

In a further embodiment of the invention the apparatus may be providedwith one or more means to facilitate mixing of the solutions used in theinvention, such as for example an enzyme solution. In this respect, andby way of example only, the apparatus may include a means for vortexingthe solution; such as an electromagnetic system that is adapted toagitate a magnetic bead. Where the apparatus includes an electromagneticmixing system the magnetic bead is preferably provided in the container(e.g., vial) in which the solution is stored in the apparatus.Alternatively the magnetic bead may be added to the solution when mixingis desired.

In a highly preferred form of the invention the mixing means is combinedwith the heating means either as a single unit or as separate units tofacilitate constant heating of the solution in an even manner. Usingsuch a mixing means avoids the possible overheating of solution closestto the heating unit while the solution is heated. Such a system willprovide a more constant heating of the solution. Alternate means formixing the solution will be known in the art and include, mechanical,physical, electrical and electromagnetic means as an example. While anymixing means may be employed in the apparatus, preferably the mixingmeans is either selected to minimize vibration of the apparatus orincorporated into the apparatus in a manner that minimize suchvibration. In this respect the mixing means may be housed on one or morevibration dampeners or the apparatus may include one or more vibrationdampeners on is base.

Where a mixing means is incorporated into the apparatus the means may beautomatically activated upon activation of the heating unit oralternatively there may be a separate activation system. Further, thespeed of the mixing may either be fixed or variable. Preferably, thereis a separate activation system for the mixing means.

The filter recess incorporated into the apparatus may be of any size orshape that facilitates filtering of a cellular suspension. Further therecess may be adapted to receive and hold at least a tube into whichcell suspension may be filtered. Preferably the recess has a conicalbase providing easy means to access the full volume of cell suspensionafter it has been filtered.

Desirably, the third recess is designed to receive a 100 μm cell filter.The third recess can accommodate a 100 μm cell filter connected to aconical tube. Preferably, the tube has area/volume graduations marked onthe side.

The apparatus may also include a set of tools required for cellharvesting. It will be appreciated by those skilled in the art that anytools necessary for cell harvesting may be included with the device.Preferably, the set of tools are sterile. As an example only, the set oftools may include a glass vessel of separation enzyme; a sterilesolution for suspension of the enzyme; a sterile nutrient solution;scalpel; forceps; syringe; medicine dropper, cell filter; wounddressings and/or spray nozzles. In a highly preferred embodiment, theset of tools are stored in a compartment formed in the first member ofthe apparatus, which is covered by the second member when not in use.

In a highly preferred embodiment, the device is used to harvest asuspension of cells and apply the cells to a recipient site in thefollowing manner.

An aliquot of sterile water is mixed with a portion of lyophilizedseparation enzyme and placed in the heating recess. The heating means isthen activated which heats the contents (i.e. the enzyme solution) ofthe container to a working temperature of between 30 and 37° C.,preferably between 33 and 37° C. and by way of example 37° C. within 2minutes and maintains the working temperature for at least 45 minutes.Once an operational temperature has been reached, a sample of tissuetaken from a donor site is placed in the enzyme solution and incubatedat the working temperature. The tissue sample is incubated for between 5to 45 minutes. Those skilled in the art would appreciate that the timetaken to achieve separation of the layers of the tissue sample isdependent on the thickness and size of the tissue sample and theincubation temperature. Once enzymatic separation of the tissue layersis achieved, the tissue sample is removed to the reservoir and thetissue layers are separated using surgical instrument/s.

A carefully measured aliquot of the second solution is then withdrawnfrom the fluid containment well by aspiration into a syringe and thenapplied to the layers. The cells between the layers of tissue arescraped off and suspended by mixing with the nutrient solution. The cellsuspension is then collected, preferably using a syringe and cannula.

The harvested suspension of cells is then passed through a cell filterlocated in the filter recess and the filtered suspension of cells iscollected into the filter recess. The reservoir may optionally be rinsedwith a further volume of the second solution and this resultingsuspension of cells also filtered and collected in the filter recess.

The filtered suspension of cells may then optionally be aspirated into asyringe and applied to the recipient site.

EXAMPLES

Further features of the present invention are more fully described inthe following non-limiting Examples. It is to be understood, however,that this detailed description is included solely for the purposes ofexemplifying the present invention. It should not be understood in anyway as a restriction on the broad description of the invention as setout above.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompositions and/or methods claimed herein are made and evaluated, andare intended to be purely exemplary of the invention and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. The present invention is moreparticularly described in the following examples which are intended asillustrative only since numerous modifications and variations thereinwill be apparent to those skilled in the art.

Example 1 Preparation of Recipient Site

To optimize the success of the skin graft, the wound was cleaned andassessed to be of the appropriate depth. Further, blood haemostasis wasestablished and the wound checked for evidence of surrounding cellulitisor infection. Techniques for preparing the area included sharpdissection, dermabrasion or laser-resurfacing.

Donor Site Biopsy

The donor site was chosen to appropriately match the recipient site. Thedonor site was infiltrated with local anaesthetic and adrenalineunderneath the skin near the subcutaneous tissue. This allowed the donorsite to be firm and aided in the taking a thin split-thickness biopsy.The dimensions of the biopsy were determined by the size of the surfacearea of the recipient site to be covered. Typically, the biopsy size hasan expansion ratio of 1:10-1:80. In this case, a biopsy size of 2 cm×2cm was taken from the donor site giving an expansion ratio of 1:60.

Cell Resurfacing Using the Rapid Technique

Treatment of the wound was carried out using the Re-Cell® RapidTechnique cell harvesting apparatus, which is explained in more detailin Example 2 below. The apparatus contained all the instruments,solutions, enzymes and dressings required for wound treatment.

The heating element was activated by depressing the “start button”.Solution (sterile water for injection) (10 ml) was transferred from thesupplied plastic vessel marked Solution A into a glass vessel containingthe separation enzyme (lyophilized trypsin) to give a finalconcentration of 0.5% trypsin. The enzyme solution was then mixedtogether, transferred to a vessel already located in the heating elementrecess and heated to 37° C.

The vessel containing Solution B (nutrient media) was transferred fromits supplied vessel into the fluid containment well.

The previously obtained tissue sample was then placed in the enzymesolution and incubated at 37° C. for between 10 to 15 minutes. Afterthis time, the tissue sample was removed from enzyme solution with apair of forceps, rinsed by dipping into the fluid containment wellcontaining Solution B and placed with the dermal side down and theepidermal side up in the reservoir.

Solution B was then aspirated from the well into a syringe and drippedfrom the syringe onto both layers of the biopsy.

The skin layers were separated using forceps. This allowed access to thezone of the dermal-epidermal junction of both surfaces. Cells werescraped from the surfaces to develop a plume of cells in the reservoir.The cells were then mixed in Solution B. The plume of cells was thendrawn up into the syringe via a 19 gauge cannula.

The supplied filter (100 μm cell filter) was mounted in the filterrecess and the plume of cells in Solution B was passed through thefilter. A further small amount of Solution B was then used to rinse thereservoir (e.g., a petri dish) and collect any remaining cells, whichwere also passed through the filter.

The resulting suspension of cells collected in the conical recess wasaspirated into a syringe and a nozzle was attached to the syringe forspraying or dripping on to the wound area.

The wound was re-checked to ensure that it was clean and free of debrisand that there was no evidence of bacterial contamination. Further, thewound was checked to determine if haemostasis had been achieved. Oncethe recipient site was ready, the suspension of cells was applied to thewound surface using the nozzle.

The wound was dressed with Surfasoft™ a woven nylon dressing, which wassupplied with the apparatus. The healing of the wound was followed upusing standard protocols for skin-graft treatment.

Example 2

The embodiment shown in FIG. 1 is directed to a Re-Cell® Rapid Techniquecell harvesting apparatus 10 for use in producing a transplantablecellular suspension of living tissue suitable for grafting to a patient.

As illustrated in FIG. 1 the apparatus includes a closure lid 12possessing a locking mechanism 14 adapted to releasably engage a baseportion 16. The locking mechanism 14 provides a means for closing theapparatus 16 when not in use. Located within the base portion 16 is afirst member 18 within which there is provided an aperture 20 in whichthere is located a vial 22 for the enzyme. Adjacent the aperture thereis provided an activation switch 24 capable of activating the heatingmeans (not shown). The first member also provides a fluid containmentwell 26 and a filter recess 28. As presented in this illustration thefilter 29 is shown located in the filter recess. Ordinarily the filteris an optional item included as a separate item in the apparatus.

The aperture 20 in the first member 18 is desirably of such a diameterthat it allows the neck of the vial 22 to protrude through and above thefirst member 18. The periphery of the aperture 20 is fitted with acollar 21 which is slightly smaller than the diameter of the body of thevial 22. Thus, when in use, the vial 22 cannot be removed from theapparatus 10 as it is held in place by the collar 21 located around theperiphery of the aperture 20.

Located adjacent to the aperture 20, fluid containment well 26 andfilter recess 28 is the second member 30 which is positioned on a seat(not shown) located within a storage compartment (not shown) within thefirst member 18. When inverted the second member 30 forms a reservoirwithin which tissue manipulations may be performed. To facilitateseparation of the second member 30 from the first member 18 an indent 32is provided in the side of a portion of the second member 30, which isof such a size that a person can lift the second member from the seat onwhich it resides in the first member 18.

Within the filter recess 28 there is located a filter 29 (providedseparately with the other components) having a mesh therein capable ofseparating cellular material of greater than 100 μm from a cellsupernatant.

FIG. 2 provides a partially exploded perspective view of the apparatus10 wherein the second member 30 is removed from the first member 18 andinverted. Inversion of the second member 30 reveals the sidewalls 32 ofthe second member 30, which form the fluid containment barrier of thereservoir and a reservoir area 34 in which tissue manipulations may beperformed.

Removal of the second member 30 from the first member 18 also reveals astorage compartment 36 in the first member 18 in which solutions andtools may be stored when the apparatus 10 is not in use. Within thestorage compartment 36 there is located a seat 38 upon which the secondmember 30 may reside. The seat 38 is preferably located around theperiphery of the storage compartment 36 at a depth beneath the surfaceof the first member 18 that is equivalent to the height of the sidewalls32 of the second member 30.

FIG. 3A provides a perspective view of the first member 18 showing thestorage compartment 36, the heater activating switch 24, the aperture20, the fluid containment well 26 and filter recess 28 formed within thefirst member. FIG. 3B provides a rear view of the first member 18showing the filter recess 28, the fluid containment well 26, the heateractivating switch 24, the aperture collar 21 and the rear wall of thestorage compartment 36. As seen in this figure the filter recess has aconical base thereby providing a means for easy access to the cellsuspension that is filtered into it. Located adjacent to the fluidcontainment well and on the opposite side of the filter containment wellthere is also provided a battery positioning member 40 which protrudestowards the base 16 of the apparatus (not shown) and provides a meansfor holding the batteries in place, which are required for activatingthe heating means.

FIG. 4 provides a perspective view of the base 16 of the apparatus 10showing the vial 22 located within a containment field 42. Between thecontainment field 42 and the vial 22 there is located a heatingcollar(s) 44, which surrounds the body of the vial. Adjacent thecontainment field there is a circuit board 46, which is held in positionby circuit board containment means 48, 50 and 52. Said circuit board 46is in electrical communications with the heating collar(s) 44 by wires54. The circuit board is also in electrical communication via wires 58with the heater activating switch (not shown). Adjacent the circuitboard 46 there is provided a battery containment means 58, which holds 4AA batteries in immovable position (not shown). The batteries are inelectrical communication with the circuit board 46 by wires 60. When thefirst member 18 is fitted to the base 16 the batteries are held in placeby the battery containment means 58, the battery positioning means 40and the base of each of the fluid containment well 26 and the filterrecess 28. Preferably the conical base of the filter recess 28 alsoprotrudes between the batteries therein providing a further means forsecuring the batteries in immovable position.

Modifications and variations of the described methods and device of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in the relevant field in which this invention resides areintended to be within the scope of the following claims.

The invention claimed is:
 1. A cell suspension for direct application toa patient, said cell suspension produced according to a methodcomprising the steps of: (a) subjecting a skin tissue sample comprisingdermis and epidermis bonded at a dermal-epidermal junction, to at leasta physical and/or chemical dissociating means capable of dissociatingcellular stratum in the skin tissue sample for sufficient time to weakencohesive bonding between tissue stratum; (b) removing the skin tissuesample from the dissociating means used in step (a) and harvesting, in areservoir containing a nutrient solution, cells from a dermal surfacefacing the dermal-epidermal junction and an epidermal surface facing thedermal-epidermal junction, wherein the nutrient solution is (i) free ofxenogenic serum, (ii) capable of maintaining viability of the cellsuntil applied to a patient and (iii) suitable for direct application toa recipient region on a patient, thereby to produce a mixed suspensionof dermal and epidermal cells; and (c) filtering the mixed suspension ofdermal and epidermal cells to remove large cellular conglomeratesgreater than 200 μm, to produce a cell suspension that is preparedperi-operatively and is directly applied to the patient; wherein themethod does not comprise the steps of pelleting and resuspending thecells; wherein the filtered cells are substantially free of cellularstratum and are suspended in the nutrient solution such that the cellsuspension is sprayable as airborne droplets.
 2. The cell suspensionaccording to claim 1, wherein the skin tissue sample is autologous tosaid patient.
 3. The cell suspension according to claim 1, wherein theskin tissue sample provides an expansion ratio of 1:10 to 1:80 in a skintreatment.
 4. The cell suspension according to claim 3, wherein the skintreatment is selected from: epidermal resurfacing; replacement afterskin loss; re-pigmentation of an area of skin; treatment of burn wounds,leukoderma, vitiligo, piebaldism, or scars; resurfacing cosmeticdermabrasion; resurfacing after laser treatment; or dermalreconstruction.
 5. The cell suspension according to claim 1, wherein theskin tissue sample is up to 2 cm×2 cm in size.
 6. The cell suspensionaccording to claim 1, wherein the disassociating means comprises anenzyme selected from the group consisting of trypsin, trypsin-EDTA,dispase, collagenase, thermolysin, pronase, hyaluronidase, pancreatin,elastase and papain.
 7. The cell suspension according to claim 1,wherein the nutrient solution is a physiological saline.
 8. The cellsuspension according to claim 1, wherein recipient region is receivingtissue grafting.
 9. The cell suspension according to claim 1, whereinrecipient region is receiving cell grafting.
 10. A cell suspensionproduced according the method comprising the steps of: (a) providing askin tissue sample from a patient, the sample comprising a dermis, anepidermis, and a dermal-epidermal junction therebetween; (b) harvestingcells from a dermal surface facing the dermal-epidermal junction and anepidermal surface facing the dermal-epidermal junction in a reservoir ofnutrient solution thereby to produce a plume of dermal and epidermalcells in the nutrient solution; and (c) filtering the plume of dermaland epidermal cells to remove cellular conglomerates greater than 200 μmto produce a cell suspension, wherein the filtered cells aresubstantially free of cellular stratum and are suspended in the nutrientsolution such that the cell suspension is sprayable as airbornedroplets, wherein the cell suspension is prepared peri-operatively anddirectly applied to the patient, and wherein the nutrient solution isfree of serum xenogenic to the patient and capable of maintainingviability of the dermal and epidermal cells.
 11. The cell suspensionaccording to claim 10, wherein the skin tissue sample provides anexpansion ratio of 1:10 to 1:80 in a skin treatment.
 12. The cellsuspension according to claim 11, wherein the skin treatment is selectedfrom: epidermal resurfacing; replacement after skin loss;re-pigmentation of an area of skin; treatment of burn wounds,leukoderma, vitiligo, piebaldism, or scars; resurfacing cosmeticdermabrasion; resurfacing after laser treatment; or dermalreconstruction.
 13. The cell suspension according to claim 10, whereinthe skin tissue sample is up to 2 cm×2 cm in size.
 14. The cellsuspension according to claim 10, wherein the nutrient solution is aphysiological saline.
 15. The cell suspension according to claim 10,wherein recipient region is receiving tissue grafting.
 16. The cellsuspension according to claim 10, wherein recipient region is receivingcell grafting.